Cortinarius subgenus Leprocybe, unexpected diversity and significant differences in species compositions between western and eastern North America.

The focus of this paper is the North American species of Cortinarius in subg. Leprocybe. Eighteen species, including twelve new ones, and two tentative (aff.) species, are delimited based on morphological and molecular data (DNA ITS-LSU sequences). Existing type specimens of species in subg. Leprocybe were also studied, and neo- or epitypes designated for C. cotoneus, C. melanotus, C. phrygianus and C. venetus to stabilize the nomenclature. In addition, to improve the infrasubgeneric classification of Leprocybe three new sections are proposed: sect. Fuscotomentosi, sect. Melanoti and sect. Squamiveneti. This study adds substantial information to the knowledge of subg. Leprocybe in North America against a background of European species. To date only two species, C. phrygianus and C. squamivenetus have been reported from both continents. Citation: Ammirati J, Liimatainen K, Bojantchev D, et al. 2021. Cortinarius subgenus Leprocybe, unexpected diversity and significant differences in species compositions between western and eastern North America. Persoonia 46: 216-239. https://doi.org/10.3767/persoonia.2021.46.08.

External quality assessment for molecular typing of Salmonella 2013-2015: performance of the European national public health reference laboratories.

We report the results of three consecutive External Quality Assessments (EQAs) for molecular subtyping of Salmonella to assess the performance of the European national public health reference laboratories (NPHRLs). The EQA included the molecular typing methods used for European enhanced surveillance of human Salmonella infections: pulsed field gel electrophoresis (PFGE), including gel analysis by the use of the software BioNumerics, and 5-locus multiple locus variable number of tandem repeat analysis (MLVA) for serovar Typhimurium. The participation in the PFGE laboratory part was higher (27/35) than in the gel analysis (19/35) and MLVA (15/35), suggestive of the need for capacity building in methods requiring specialized equipment (MLVA) or software (gel analysis). The majority (25/27) of the participating NPHRLs produced inter-laboratory comparable PFGE gel(s). Two laboratories continued to produce low-quality gels and should have additional technical assistance in the future. In particular, two gel quality evaluation parameters, measuring "image acquisition and running conditions" and "bands", were identified to cause gel quality problems throughout the EQAs. Despite the high number of laboratories participating in the PFGE laboratory part, the participation in gel analysis was low, although increasing. In the MLVA part, the NPHRLs correctly assigned 96% (405/420) allelic profiles according to the nomenclature. In conclusion, the EQAs identified critical parameters for unsuccessful performance and helped to offer assistance to those laboratories that needed it most. The assessments supported the development of quality in molecular typing and promoted the harmonization of subtyping methods used for EU/EEA-wide surveillance of human Salmonella infections.

Cortinarius section Bicolores and section Saturnini (Basidiomycota, Agaricales), a morphogenetic overview of European and North American species.

Cortinarius is the largest genus of ectomycorrhizal fungi worldwide. Recent molecular studies have shown high levels of morphological homoplasy within the genus. Importantly, DNA phylogenies can reveal characteristics that have been either over- or underemphasized in taxonomic studies. Here we sequenced and phylogenetically analysed a large set of pan-European and North American collections taxonomically studied and placed in Cortinarius sect. Bicolores and sect. Saturnini, according to traditional morpho-anatomical criteria. Our goal was to circumscribe the evolutionary boundaries of the two sections, to stabilize both the limits and nomenclature of relevant species, and to identify described taxa which, according to our current understanding, belong to other lineages. Our analysis resolves two clades: /Bicolores, including 12 species, one of which is new to science, and /Saturnini, including 6 species. Fifteen binomials, traditionally treated in these two sections based on morphology, do not belong to the above two phylogenetic clades. Instead, six of these latter are clearly placed in other clades that represent sect. Bovini, sect. Sciophylli, sect. Duracini and sect. Brunneotincti. The presence or absence of blue pigments and the detection of specific odours emerge as clearly misleading taxonomic features, but more surprisingly, spore size and ecology can be misleading as well. A total of 63 type specimens were sequenced, 4 neotypes and 2 epitypes are proposed here, and 1 new combination is made.

Food-borne diseases associated with frozen berries consumption: a historical perspective, European Union, 1983 to 2013.

Epidemiological investigations of outbreaks of hepatitis A virus (HAV) and norovirus (NoV) infections in the European Union/European Economic Area (EU/EEA) in the last five years have highlighted frozen berries as a vehicle of infection. Given the increasing berry consumption in the EU over the last decades, we undertook a review of the existing evidence to assess the potential scale of threat associated with this product. We searched the literature and four restricted-access online platforms for outbreak/contamination events associated with consumption of frozen berries. We performed an evaluation of the sources to identify areas for improvement. The review revealed 32 independent events (i.e. outbreak, food contamination) in the period 1983­2013, of which 26 were reported after 2004. The identified pathogens were NoV, HAV and Shigella sonnei. NoV was the most common and implicated in 27 events with over 15,000 cases reported. A capture­recapture analysis was performed including three overlapping sources for the period 2005­2013. The study estimated that the event-ascertainment was 62%. Consumption of frozen berries is associated with increasing reports of NoV and HAV outbreaks and contamination events, particularly after 2003. A review of the risks associated with this product is required to inform future prevention strategies. Better integration of the available communication platforms and databases should be sought at EU/EEA level to improve monitoring, prevention and control of food-borne-related events.

Risk ranking of pathogens in ready-to-eat unprocessed foods of non-animal origin (FoNAO) in the EU: initial evaluation using outbreak data (2007-2011).

Foods of non-animal origin (FoNAO) are consumed in a variety of forms, being a major component of almost all meals. These food types have the potential to be associated with large outbreaks as seen in 2011 associated with VTEC O104. In order to identify and rank specific food/pathogen combinations most often linked to human cases originating from FoNAO in the EU, a semi-quantitative model was developed using seven criteria: strength of associations between food and pathogen based on the foodborne outbreak data from EU Zoonoses Monitoring (2007-2011), incidence of illness, burden of disease, dose-response relationship, consumption, prevalence of contamination and pathogen growth potential during shelf life. The top ranking food/pathogen combination was Salmonella spp. and leafy greens eaten raw followed by (in equal rank) Salmonella spp. and bulb and stem vegetables, Salmonella spp. and tomatoes, Salmonella spp. and melons, and pathogenic Escherichia coli and fresh pods, legumes or grains. Despite the inherent assumptions and limitations, this risk model is considered a tool for risk managers, as it allows ranking of food/pathogen combinations most often linked to foodborne human cases originating from FoNAO in the EU. Efforts to collect additional data even in the absence of reported outbreaks as well as to enhance the quality of the EU-specific data, which was used as input for all the model criteria, will allow the improvement of the model outputs. Furthermore, it is recommended that harmonised terminology be applied to the categorisation of foods collected for different reasons, e.g. monitoring, surveillance, outbreak investigation and consumption. In addition, to assist future microbiological risk assessments, consideration should be given to the collection of additional information on how food has been processed, stored and prepared as part of the above data collection exercises.

The largest type study of Agaricales species to date: bringing identification and nomenclature of Phlegmacium (Cortinarius) into the DNA era.

Cortinarius is a species-rich and morphologically challenging genus with a cosmopolitan distribution. Many names have not been used consistently and in some instances the same species has been described two or more times under separate names. This study focuses on subg. Phlegmacium as traditionally defined and includes species from boreal and temperate areas of the northern hemisphere. Our goals for this project were to: i) study type material to determine which species already have been described; ii) stabilize the use of Friesian and other older names by choosing a neo- or epitype; iii) describe new species that were discovered during the process of studying specimens; and iv) establish an accurate ITS barcoding database for Phlegmacium species. A total of 236 types representing 154 species were studied. Of these 114 species are described only once whereas 40 species had one ore more synonyms. Of the names studied only 61 were currently represented in GenBank. Neotypes are proposed for 21 species, and epitypes are designated for three species. In addition, 20 new species are described and six new combinations made. As a consequence ITS barcodes for 175 Cortinarius species are released.

Laboratory preparedness for detection and monitoring of Shiga toxin 2-producing Escherichia coli O104:H4 in Europe and response to the 2011 outbreak.

A hybrid strain of enteroaggregative and Shiga toxin 2-producing Escherichia coli (EAEC-STEC) serotype O104:H4 strain caused a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in 2011 in Europe. Two surveys were performed in the European Union (EU) and European Economic Area (EEA) countries to assess their laboratory capabilities to detect and characterise this previously uncommon STEC strain. Prior to the outbreak, 11 of the 32 countries in this survey had capacity at national reference laboratory (NRL) level for epidemic case confirmation according to the EU definition. During the outbreak, at primary diagnostic level, nine countries reported that clinical microbiology laboratories routinely used Shiga toxin detection assays suitable for diagnosis of infections with EAEC-STEC O104:H4, while 14 countries had NRL capacity to confirm epidemic cases. Six months after the outbreak, 22 countries reported NRL capacity to confirm such cases following initiatives taken by NRLs and the European Centre for Disease Prevention and Control (ECDC) Food- and Waterborne Disease and Zoonoses laboratory network. These data highlight the challenge of detection and confirmation of epidemic infections caused by atypical STEC strains and the benefits of coordinated EU laboratory networks to strengthen capabilities in response to a major outbreak.

Raw grated beetroot linked to several outbreaks of sudden-onset gastrointestinal illness, Finland 2010.

In 2010, 7/44 (16%) reported foodborne outbreaks in Finland were linked with raw beetroot consumption. We reviewed data from the national outbreak registry in order to hypothesize the aetiology of illness and to prevent further outbreaks. In the seven outbreaks, 124 cases among 623 respondents were identified. Consumption of raw beetroot was strongly associated with gastrointestinal illness (relative risk 8∙99, 95% confidence interval 6∙06-13∙35). The illness was characterized by sudden onset of gastrointestinal symptoms; the median incubation time was 40 min and duration of illness 5 h. No common foodborne pathogens or toxins were found in either clinical or beetroot samples, but all tested beetroot samples were of poor quality according to total bacterial counts. Beta-haemolytic Pseudomonas fluorescens was detected in several beetroot samples but its effect on human health is unknown. No outbreaks were reported after the Finnish Food Safety Authority Evira advised against serving raw beetroot in institutional canteens.

A phase I, dose-escalation study of TB-403, a monoclonal antibody directed against PlGF, in patients with advanced solid tumours.

BACKGROUND: TB-403 (RO 5323441), a humanised monoclonal antibody, is a novel antiangiogenesis agent directed against placental growth factor. The safety, pharmacokinetics (PK), and antitumour activity of TB-403 were assessed in a phase I, dose-escalation study in patients with advanced solid tumours. METHODS: Patients in sequential dose groups received either weekly doses of 1.25, 5.0, or 10 mg kg(-1) or doses of 20 or 30 mg kg(-1) every third week. RESULTS: Twenty-three patients were enrolled and received TB-403. The most common adverse events (AEs) were fatigue, constipation, pyrexia, dyspnoea, and nausea. One serious AE, a lung embolus in a patient with non-small cell lung cancer treated with 10 mg kg(-1) weekly, was deemed possibly related to TB-403. No dose-limiting toxicities were observed, and a maximum-tolerated dose was not reached. The PK parameters were dose linear and the terminal half-life values ranged from 9 to 14 days. Six patients exhibited stable disease for at least 8 weeks. Two patients, (oesophageal squamous cell carcinoma and pancreatic adenocarcinoma) both treated with 5 mg kg(-1) weekly, remained stable for 12 months. CONCLUSION: TB-403 treatment in this patient population is well tolerated, with a safety profile distinct from that of vascular endothelial growth factor-axis inhibitors.

Multiple norovirus outbreaks linked to imported frozen raspberries.

In 2009, the number of foodborne norovirus outbreaks in Finland seemed markedly high, and many outbreaks seemed to be linked to imported frozen raspberries. We reviewed the data regarding all notified foodborne outbreaks in 2009 in Finland in order to assess the magnitude of the problem and to summarize the information on raspberry-linked outbreaks. Between March and August, 13 norovirus outbreaks affecting about 900 people could be linked to imported frozen raspberries. Two raspberry samples corresponding to two batches of raspberries were positive for norovirus. These two batches proved to have been the likely source in six of the 13 outbreaks. Analytical studies had not been conducted for six outbreaks, and virological test results were inconclusive in two. However, combining epidemiological and microbiological methods often enabled finding the source, as exemplified in investigation of a large school outbreak. To ensure prompt control measures in similar situations in the future, both aspects of outbreak investigations should be strengthened.

A nationwide outbreak of Salmonella bovismorbificans associated with sprouted alfalfa seeds in Finland, 2009.

Salmonella enterica serotype Bovismorbificans is a rare serotype in Finland. In June 2009, a nationwide outbreak of S. Bovismorbificans infections occurred, and 42 clinical isolates were identified. We conducted a case-control study enrolling 28 cases and 48 matched controls, and found ready-to-eat alfalfa sprouts associated with the infection (odds ratio = 35.2, 95% confidence interval 2.8-435). The sprouts were traced back to a domestic producer, with the seeds originating in Italy. Although finding microbiological evidence for sprouts as a source of this outbreak was very challenging, S. Bovismorbificans was finally found in sprouts germinated in the laboratory, and in soaking and rinse waters during the germination process. The pulsed field gel electrophoresis showed that these isolates were identical to the human outbreak isolates. Before sampling, it is important to mix the seeds well and to take several large-size samples from different seed lots. Instead of analysing seeds, the analysis should be targeted to soaking or rinse water samples and to the sprouts germinated in a laboratory. Accordingly, the sprout producers should only buy seeds that have been analysed for Salmonella. The producers have to include Salmonella testing in their internal quality control to ensure that Salmonella is absent from sprouts placed on the market during their shelf-life. In order to reduce the health hazard caused by sprouts, an effective and safe decontamination procedure should also be developed for the seeds.

Detection of human norovirus from frozen raspberries in a cluster of gastroenteritis outbreaks.

We describe a cluster of norovirus outbreaks affecting about 200 people in Southern Finland in September and October 2009. All outbreaks occurred after consumption of imported raspberries from the same batch intended for the catering sector. Human norovirus genotype GI.4 was found in frozen raspberries. The berries were served in toppings of cakes in separate catering settings or mixed in curd cheese as a snack for children in a daycare center. The relative risk for consumption of the berry dish was 3.0 (p

Botulism and hot-smoked whitefish: a family cluster of type E botulism in France, September 2009.

A family cluster of three cases of type E botulism were identified in south-east France in September 2009. The suspected food source of infection was a vacuum packed hot-smoked whitefish of Canadian origin purchased by the family during a visit to Finland and consumed several weeks later in France on the day prior to symptom onset. No leftover fish was available to confirm this hypothesis. Vacuum packed hot-smoked whitefish has previously been associated with cases of type E botulism in multiple countries, including Finland, Germany, the United States and Israel.

Yersinia pseudotuberculosis causing a large outbreak associated with carrots in Finland, 2006.

A large outbreak of Yersinia pseudotuberculosis O:1 infection affected over 400 children from 23 schools and 5 day-care centres in two municipalities in southern Finland in August-September, 2006. A retrospective cohort study conducted in a large school centre showed that the outbreak was strongly associated with the consumption of grated carrots served at a school lunch. The risk of illness increased with the amount of carrots eaten. Poor quality carrots grown the previous year had been delivered to the school kitchens in the two municipalities affected. In the patients' samples and in the environmental samples collected from the carrot distributor's storage facility, identical serotypes and genotypes of Y. pseudotuberculosis were found, but the original source and the mechanism of the contamination of the carrots remained unclear. Outbreaks of Y. pseudotuberculosis linked to fresh produce have been detected repeatedly in Finland. To prevent future outbreaks, instructions in improved hygiene practices on the handling of raw carrots have been issued to farmers, vegetable-processing plants and institutional kitchens.

Characterisation of non-pathogenic Yersinia pseudotuberculosis-like strains isolated from food and environmental samples.

Non-pathogenic Yersinia pseudotuberculosis-like strains were recovered from Finnish food and environmental samples. These strains could not be differentiated from Y. pseudotuberculosis strains using API 20E or other phenotypical tests. However, all of the strains were inv-, and virF-negative with polymerase chain reaction (PCR), while all Y. pseudotuberculosis strains used as controls were inv-positive and fresh Y. pseudotuberculosis strains were also virF-positive, indicating that the Y. pseudotuberculosis-like strains were non-pathogenic. Using pulsed-field gel electrophoresis (PFGE) with NotI enzyme and ribotyping with EcoRI and HindIII enzymes, the Y. pseudotuberculosis-like strains, which grouped genetically together, could be differentiated from true Y. pseudotuberculosis strains and from strains belonging to other sucrose-negative Yersinia species. In addition, the O-antigen gene cluster of one Y. pseudotuberculosis-like strain was characterized, and it differed from those of known Y. pseudotuberculosis serotypes. This study demonstrates that identification of Y. pseudotuberculosis from food and environmental sources using solely biochemical reactions can be incorrect, and when a strain cannot be serotyped to known Y. pseudotuberculosis serotypes, the pathogenic potential of isolates should be determined.

Multiple norovirus outbreaks among workplace canteen users in Finland, July 2006.

Multiple gastroenteritis outbreaks occurred between 25 and 31 July 2006 in 10 workplace canteens in south-western Finland. One vegetable processing plant provided raw vegetables to all the canteens. We conducted cohort studies in the three most visited canteens and environmental investigations in the kitchens and the plant. Patients' stools, food, water and environmental samples were tested for enteric bacteria and viruses. Of the three canteens, 150/273 respondents (response rate 82%) had gastroenteritis. Consumption of mixed raw vegetables was significantly associated with the illness but no single vegetable explains the outbreak. An identical norovirus GII.1 genotype was detected from all genotyped patient samples. Water, food, and environmental samples were negative for norovirus. The facilities had appropriate hygienic conditions and no staff member had gastroenteritis prior to the outbreak. Tracing back the vegetables to the farm level proved unsuccessful. This was the largest foodborne norovirus outbreak in Finland.

Distribution of virF/lcrF-positive Yersinia pseudotuberculosis serotype O:3 at farm level.

The distribution and persistence of pathogenic, virF/lcrF-positive Yersinia pseudotuberculosis were investigated in pigs and in the pig house environment during rearing to determine possible contamination routes of early infections. Based on Y. pseudotuberculosis-positive tonsils of slaughter pigs in our previous study, Y. pseudotuberculosis-positive animals were traced back to the farms. Eight farms were visited from 6-10 months later, and a total of 155 pooled and six individual faecal samples from pigs and 116 pooled environmental samples were collected for analysis by different culture methods. Four of the eight farms were found to be Y. pseudotuberculosis-positive. All positive faecal samples were obtained from fattening pigs, with prevalence varying from 5% to 71% on positive farms. Sows, boars and suckling piglets were Y. pseudotuberculosis-negative on all farms. Most Y. pseudotuberculosis-positive farms (three of four) were on a one-site production system, which had a higher prevalence of Y. pseudotuberculosis (5-26%) among fattening pigs than the all-in, all-out system (1-5%). All Y. pseudotuberculosis isolates belonged to serotype O:3 and carried the virF/lcrF gene on the virulence plasmid. Biotypes 2 and 3 were involved, the latter in one isolate and not being previously reported in pigs. Altogether 53 isolates from 16 positive samples were characterized with pulsed-field gel electrophoresis (PFGE). Using SpeI, NotI and XbaI enzymes, four, three and two different PFGE patterns were obtained respectively. A total of nine different genotypes were identified when the profiles of the enzymes were combined. The most common genotypes were gIV, found on three, and gXII, found on two of the four Y. pseudotuberculosis-positive farms. The same genotypes previously detected in pig tonsils were present in pig faeces from the same farm, indicating that some Y. pseudotuberculosis strains can persist in the pig house environment.

Different Yersinia enterocolitica 4:O3 genotypes found in pig tonsils in Southern Germany and Finland.

The distribution of different genotypes of Y. enterocolitica 4:O3 strains recovered from pig tonsils in Southern Germany and Finland in 1999-2000 was investigated. A total of 96 and 207 Y. enterococolitica 4:O3 isolates recovered from 47 and 66 tonsils of finishing pigs in Germany and Finland, respectively, were characterised with PFGE using NotI enzyme. In all, 39 different NotI profiles were obtained, only one of which, NB1, was found in both Germany and Finland. All strains were further characterised with ApaI and XhoI enzymes. When the 54 German and 74 Finnish strains were characterised with all three enzymes, 51 genotypes were obtained. The 23 genotypes found in German strains differed from the 28 found in Finnish strains. These results indicate that Y. enterocolitica 4:O3 genotypes have a differential geographical distribution and thus can be used in epidemiological studies.